CHROMOGENIC SUBSTRATE TECHNOLOGY

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CHROMOGENIC SUBSTRATE TECHNOLOGY

• Giovanni Russi

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What is a chromogenic substrate?

• A peptide linked to a chromophore
• The peptide is formed by 3-5 residues
• The chromophore is p-nitroaniline (p-NA)
• The residues can be natural amino acids or
  chemically modified amino acids
• The sequence of the residues mimics the sequence
  of the natural substrate
• The hydrolysis of the substrate causes the
  release of pNA (yellow colored compound)

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Chemical structure
Prothrombin, the natural substrate of FXa, is
cleaved by FXa at two positions, each proceeded
by the same four amino acid sequence. FXa
activity can be determined by the chromogenic
substrate S-2222 which is composed of the same
amino acids coupled to a chromophore
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Chemical structure
Blocking group
Chromophore
Residues
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Chemical structure

• S-2222 a substrate specific for FXa

Bz-Ile-Glu(g-OR)-Arg-pNA
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Enzymes

• Proteins that catalyze chemical reactions
• They exerts its catalytic activity upon
  substrates
• Proteolytic enzymes act on their natural
  substrates, proteins, by hydrolyzing one or more
  peptide bond(s)
• The hydrolyzing process is usually highly
  specific as only peptide bonds adjacent to
  certain amino acids are cleaved

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Classes of proteases
Asp not always present
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Serine proteases

• Two groups Trypsins and Subtilisin

In bacteria only
Trypsin Chymotrypsin Elastase Tryptase Blood coag
factors
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Trypsins

• The Trypsin family is classified according to
  the type of amino acid (a.a.) that occurs at the
  preferred cleavage site
• Enzyme Cleavage site
• Elastase hydrophobic a.a.
• Chymotrypsin aromatic a.a.
• Others basic a.a. (Arg or Lys)

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The catalytic site

• The reaction is the result of the interaction
  between the substrate and the catalytic site
• The catalytic site is known as the catalytic
  triad

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The proteolytic reaction
Formation of an acyl-enzyme intermediate
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The proteolytic reaction
Hydrolysis of the acyl-enzyme intermediate
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Enzyme kinetics
K1 K3
E S ES E P
K2
S
V Vmax
S Km
k2 k3
Km
k1
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Enzyme kinetics

• kcat is the turnover number and corresponds to
  k3. It is the maximal number of substrate
  molecules that can be converted to product per
  time unit
• Km corresponds to the concentration of substrate
  which gives a reaction rate of Vmax/2.

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Enzyme units

• The enzymatic activity is defined in two ways
• By comparison with the activity of a standard
  preparation, where the units are defined by WHO,
  NIH etc
• By measuring the amount of substrate split, or
  the product formed per time unit

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Enzyme activity calculation

• 1nkat 1 x 10-9 mol product released per sec
• pNA has a molar absorptivity of 9600 mol-1 L
• A general chromogenic method can be summarized as
  follows
• Compound Volume (mL)
• Buffer v1
• Sample v2
• Substrate v3
• A) Initial rate method reading at 405 nm and
  determination of DA/min
• B) Acid stopped method incubation (t) and
  addition of Acetic acid
• Acetic acid v4
• nkat/mL 1.74 x V/v2 x DA/min (Initial rate
  method)
• nkat/mL 1.74 x V/(v2 x t) x A (Acid stopped
  method)

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Historical background

• The application of chromogenic substrates in
  hemostasis began in the early 1970s
• BAPNA was the first chromogenic substrate for
  serine proteases but with poor selectivity
• S-2160 was the first chromogenic thrombin
  substrate
• Among the 500 pNA peptides synthesized, 24 have
  been found having the best specificity and
  reactivity towards the enzymes studied
• Now, our product range covers quite extensively
  the multiple needs of the customers

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Application of the chromogenic technology

• Antithrombin
• Heparin
• Protein C

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Antithrombin

• Antithrombin is the major thrombin inhibitor,
  accounting for approximately 80 of the thrombin
  inhibitory activity in plasma.

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Thrombin inhibition

• Inhibitors - Antithrombin -
  a2-macroglobulin - Trypsin inhibitor -
  Heparin cofactor II
• Thrombomodulin - Turns thrombin into a
  protein C activator

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Antithrombin - the protein

• 58 KDa single-chain plasma glycoprotein
• Synthesised in the liver
• Plasma concentration 150 mg/ml (2.5 mM)
• Half-life 3 days

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Antithrombin - the inhibitor

• Antithrombin inhibits thrombin, FIXa, FXa, FXIa,
  FXIIa and the complement enzyme C1.
• Antithrombin forms a 11 complex with the
  inhibited protease.
• The inhibition is enhanced by heparan sulphate, a
  heparin like substance on the endothelial cells,
  lining the blood vessels.
• Binding of heparan sulphate to antithrombin
  induces a conformational change in the
  antithrombin molecule at the reaction site. This
  facilitates its reaction with the enzyme.

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Thrombin inhibition catalysed by heparin
P
AT
H
P
AT
R
H
R
IIa
IIa
P
P
AT
H
AT
H
R
R
IIa
IIa
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FXa inhibition catalysed by heparin
P
AT
H
P
AT
R
H
R
Xa
Xa
P
P
AT
H
AT
H
R
R
Xa
Xa
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Antithrombin monitoring
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Antithrombin monitoringchromogenic activity
assays
Chromogenic heparin cofactor activity assays -
The sample is incubated with heparin and an
excess amount of thrombin or FXa. The residual
thrombin or FXa then cleaves a chromogenic
substrate
AT Heparin
ATHeparin
ATHeparin FXa(excess)
AT-FXa- Heparin FXa(residual)
FXa(residual)
Chromogenic substrate
Peptide pNA
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Antithrombin anti-FXa assay
Sample/Standard dilution 25 ml
sample 3000 ml saline Procedure Vol
umes Diluted sample/standard 50 ml Factor Xa
(2.9 nkat/ml in Hep Buffer) 50 ml Incubate at
37C 90 sec S-2765 (0.8 mg/ml) 50 ml Read
DA/min at 405 nm for rate method or add 50 ml
Acetic acid after 90 sec incubation for end-point
method.
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Antithrombin anti-FXa assay
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Heparin

• Heparin is a heterogeneous mixture of unbranced
  polysaccharide chains
• Alternating monosaccharide units of L-iduronic
  acid and D-glucosamine
• The molecule size in the natural extract is 2 to
  40 Kda
• One third of the polysaccharide chains contain a
  specific antithrombin binding pentasaccharide
  sequence

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Heparin
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Heparin

• Mechanism of action
• Heparin exerts parts of its anticoagulant
  activity through interaction with antithrombin
• Antithrombin binds specifically to a
  pentasaccharide in heparin
• Binding to heparin induces a conformational
  change in the antithrombin, which accelerate the
  enzyme inhibition

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Heparin chromogenic methods

• Anti-FXa activity

AT Heparin
ATHeparin
ATHeparin FXa(excess)
AT-FXa-Heparin FXa(residual)
FXa(residual)
Chromogenic substrate
Peptide pNA
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Heparin chromogenic methods

• Anti-FIIa activity

AT Heparin
ATHeparin
ATHeparin FIIa(excess)
Heparin-AT-FIIa FIIa(residual)
FIIa(residual)
Chromogenic substrate
Peptide pNA
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Heparin measurements
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Heparin one-stage and two-stage

• One-stage (CM Heparin)
• Sample/standard Dilution
• 100 ml sample
• 300 ml water
• Diluted sample 50 ml
• S-2732 (3 mg/ml) 50 ml
• FXa (7 nkat/ml) 50 ml
• Incubate at 37C 120 sec
• Acetic acid 50 ml
• Read at 405 nm

• Two-stage (CT Heparin)
• Sample/Standard Dilution
• 100 ml sample
• 100 ml AT (1 IU/ml)
• 800 ml Buffer
• Diluted sample 200 ml
• FXa (7.1 nkat/ml) 100 ml
• Incubate at 37C 30 sec
• S-2222 (0.75 mg/ml) 200 ml
• Incubate at 37C 180 sec
• Acetic acid 300 ml
• Read at 405 nm

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Protein C in the coagulation system
Extrinsic pathway

FX
TF FVIIa
Prothrombin
Intrinsic pathway
PL, Ca2
FIXa
FVIIIa
PL, Ca2
FXa
FVa
Protein S FV
Thrombin
APC
Thrombo-modulin
Fibrinogen Fibrin
Protein C
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Protein C - an anticoagulant

• Vitamin K dependent proteins
• Anticoagulants protein C and protein S
• Procoagulants Factor II, VII, IX and X
• Synthesised in the liver
• Glu Gla (glutamic acid residues are
  converted to gamma-carboxyglutamic acid)
• The Gla domain binds calcium ions which form a
  bridge to the phospholipid surfaces on platelets
  and endothelial cells.

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Protein C-the structure

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Protein C- the function

• Protein C inhibits coagulation through
  inactivation of FVIIIa and FVa
• Protein C potentiates the fibrinolytic system by
  inhibiting PAI-1, the major inhibitor of
  fibrinolysis.

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Protein C activation

• Protein C is activated (to APC) by thrombin.
• A small peptide is removed.
• Activation by thrombin alone is slow. The complex
  thrombin-thrombomodulin activates protein C 20
  000 times faster.

APC
PC
TM
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Protein C activation

• Thrombomodulin (TM) is a membrane protein present
  on the endothelium.
• TM has the following effects on thrombin
• Increases the rate by which thrombin activates
  protein C
• Removes the procoagulant properties of thrombin
• Accelerates the thrombin-antithrombin reaction

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APC cofactors

• APC has two known cofactors Protein S and Factor
  V.
• Protein S
• Protein S enhances binding of APC to the
  phospholipid of platelets and endothelial cells.
• Only free protein S has a APC cofactor function.
  60 of protein S is bound to C4bBP.
• Factor V
• Factor V together with Protein S makes APC
  degrade FVIIIa and FVa more effectively.

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Protein C assays - principlesChromogenic assays

• Chromogenic assays
• Utilise specific protein C activator (Protac)
  for the activation of protein C
• The activated protein C cleaves a chromogenic
  substrate.

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Coamatic Protein C - the principle

Protac
Protein C
APC
APC
S-2366
Peptide pNA
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Back to the chromogenic substrates.
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Product range
S-2222TM ? Factor Xa S-2238TM ?
Thrombin S-2251TM ? Plasmin and
Streptokinase-activated Plasminogen S-2266TM ?
Glandular Kallikrein and Factor XIa S-2288TM ?
t-PA other proteases S-2302TM ? plasma
kallikrein Factor XIa Factor XIIa S-2314TM ?
C1s S-2366TM ? activated protein C factor
XIa S-2390TM ? Plasmin S-2403TM ? Plasmin and
Streptokinase-activated Plasminogen S-2423TM ?
Endotoxin determination S-2444TM ?
Urokinase S-2484TM ? Granulocyte
elastase S-2586TM ? Chymotrypsin S-2765TM ?
Factor Xa S-2772TM ? Factor Xa
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Beyond the chemical synthesis

• Scientific support
• Well characterized substrates, i.e. kinetic
  tables
• Research Methods
• New Methods
• Pharmacopoeia abstracts
• Complementary products Bioreagents

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Substrate selectivity
The Chromogenix catalogue includes a section
which shows the cross-reactivity of the
substrates with the different enzymes tested
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Kinetic data
The Chromogenix catalogue includes a section
which shows the kinetic data of the substrates
toward the different enzymes tested
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The 25 mg Substrates

• The substrates are packaged in vials containing
  25 mg lyophilized substrate and mannitol as a
  bulking agent
• The insert sheet is in three languages and
  contains the following information
• Chemical Name Kinetic data
• Formula Standardization
• Solubility Applications
• Stability References

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The 25 mg Substrates
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The bulk substrates

• All the substrates present in the catalogue can
  be supplied as bulk material if the amount
  required is very high (grams)
• The bulk consists of a powder composed by the
  substrate, without the addition of mannitol
• The bulk is provided in vials, with a vial label
  and a certificate of analysis

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Substrates Applications The Chromogenix Kits

• Coamatic AT... S-2765
• Coamatic AT 400... S-2772
• Coamatic LR AT. S-2772
• Coamatic Protein C... S-2366
• Coaset Factor VII... S-2765
• Coamatic Factor VIII.. S-2765
• Coatest Factor VIII. S-2222
• Coatest Soluble Fibrin... S-2403
• Coamatic Heparin.. S-2732
• Coatest LMW Heparin/Heparin... S-2732
• Coatest Heparin. S-2222
• Coatest PAI S-2403
• Coaset t-PA S-2251
• Coamatic Plasmin Inhibitor.. S-2403
• Coamatic Plasminogen. S-2403

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Substrates Applications The Chromogenix
Research Methods

• Proteolytic Activity. S-2288
• Urokinase S-2444
• Factor X.. S-2765
• t-PA. S-2288
• Prekallikrein activator (PKA) S-2302
• Kallikrein-like activity. S-2302
• Kallikrein inhibitor.. S-2302
• Prekallikrein... S-2302
• Urine Kallikrein.. S-2266
• Granulocyte Elastase... S-2484
• Trypsin S-2222
• Chymotrypsin. S-2586
• Antithrombin (FIIa). S-2238
• Heparin (FIIa).. S-2238

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Substrates Applications The Chromogenix New
Methods

• Prothrombin Activity S-2238
• Hirudin. S-2366

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Substrates Applications Chromogenic methods in
quality control -European and U.S. Pharmacopoeia

• The European Pharmacopoeia
• S-2238 ? Antithrombin potency
• LMW-heparin activity (Anti-FIIa) Heparin
  in factor concentrates
• S-2765 ? LMW-heparin activity (Anti-FXa)
• S-2302 ? PKA in albumin and IgG

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Substrates Applications Chromogenic methods in
quality control -European and U.S. Pharmacopoeia

• The U.S. Pharmacopoeia
• S-2222 ? Heparin (anti-FXa activity)
• S-2288 ? Alteplase
• FDA recommendations
• S-2302 ? PKA in albumin and IgG